Under conditions of oxidative stress it is well known that the bioavailability of nitric oxide (NO) is known to be significantly reduced. This process is in part due to the combination of NO with superoxide radicals to form peroxynitrite (ONOO(-)). While this process inactivates NO per se, it is not certain to which extent this process may also further impair ongoing NO production. Given the pivotal role of arginine availability for NO synthesis we determined the impact of ONOO(-) on endothelial arginine transport and intracellular arginine metabolism. Peroxynitrite reduced endothelial [(3)H]-L-arginine transport and increased the rate of arginine efflux in a concentration-dependent manner (both p<0.05). In conjunction, exposure to ONOO(-) significantly reduced the intracellular concentration of L-arginine, N(G)-hydroxy-L-arginine (an intermediate of NO biosynthesis) and citrulline by 46%, 45% and 60% respectively (all p<0.05), while asymmetric dimethyl arginine (ADMA) levels rose by 180% (p<0.05). ONOO(-) exposure did not alter the cellular distribution of the principal L-arginine transporter, CAT1, rather the effect on CAT1 activity appeared to be mediated by protein nitrosation. Conclusion Peroxynitrite negatively influences NO production by combined effects on arginine uptake and efflux, most likely due to a nitrosative action of ONOO(-) on CAT-1.